Multiplex detection and identification of proteins on a PVDF membrane blocked with a synthetic polymer-based reagent.
2-DE is one of the most powerful methods for analyzing proteins expressed in cells and tissues. Immunodetection of proteins blotted on a polymer membrane is the method of choice for detecting specific proteins in 2-D gels. To precisely locate spots of immunoreactive proteins in 2-D gels, both dye staining and immunodetection were performed on the same PVDF membrane. Prior to immunodetection, nonspecific adsorption of the antibodies to the membrane was blocked with a synthetic polymer-based reagent (N-102) after protein transfer. The protein was then stained with colloidal gold or CBB followed by protein spot identification by LC-MS. Described herein is a method for multiplex analysis of proteins transferred to a PVDF membrane. Proteins that were phosphorylated at tyrosine in the phosphoproteome of rice callus or human ovarian cancer cells were detected by immunoblotting and subsequently identified with high precision.
Kawasaki
Hiroshi
H
Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, Yokohama, Japan. kawasaki@yokohama_cu.ac.jp
Okayama
Akiko
A
Iwafune
Yuko
Y
Yahagi
Shota
S
Arakawa
Noriaki
N
Hirano
Hisashi
H
D000818:Animals; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D007118:Immunoassay; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D012275:Oryza / Q000737:chemistry; D010051:Ovarian Neoplasms; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D010940:Plant Proteins / Q000032:analysis; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D005465:Fluorocarbon Polymers
D007202:Indicators and Reagents; D008567:Membranes, Artificial; D019000:Phosphotyrosine; D010940:Plant Proteins; D011145:Polyvinyls; D011506:Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers